HU193696B - Process for preparing new antibiotic complex sb 22484 and pharmaceutically acceptable salts thereof - Google Patents

Abstract

The present invention concerns a new antibiotic substance arbitrarily designated as antibiotic SB 22484, a process for its production by culturing Streptomyces sp. NRRL 15496 or a producing variant or mutant thereof, and the use of the new antibiotic substance in the treatment of infectious diseases involving microorganisms susceptible to this antibiotic substance such as Neisseriae strains.

Description

The present invention relates to a novel antibiotic SB 22484 for use in the treatment of Streptomyces sp. NRRL 15496 or a production variant or mutant thereof.

To simplify the description, when dealing with the biological activity of the compounds of the invention, the term "antibiotic SB 22484 for the §B 22484 antibiotic complex - consisting essentially of four major components, antibiotics SB 22484, factors 1, 2, 3 and 4 - and pharmaceutically acceptable salts thereof.

The term "lower alkyl alkanoyl esters" refers to (C 1 -C 6) alkyl (C 2 -C 6) alkanoyl esters such as ethyl acetate, propyl acetate, butyl acetate, ethyl propionate, butyl propionate, butyl amyl, etc.

The term "water-immiscible lower alkyl ketones" refers to C5-C10 ketones that are readily miscible with water, such as 2-pentanone, 2-hexanone, 3-hexanone, 2-heptanone, 3-methyl-2-butanone, and the

3-methyl-2-pentanone.

The term "halogenated lower hydrocarbons" refers to polyhalogenated C 1-4 hydrocarbons, e.g. methylene chloride, chloroform, dichloroethane, etc. It applies. The term "higher alkanols" is straight or branched linear or cyclic

Refers to C 4 -C 7 alkanols which are readily miscible with water; such as butanol, 2-methylpropanol, 2-methyl-2-propanol, pentanol, 2-methylbutanol, 2-methyl-2-butanol, cyclohexanol and the like.

The antibiotic of the present invention is characterized by a rather specific antimicrobial spectrum when it is active against Neisseria strains, rather active against Streptococcus, Ureaplasma, Mycoplasma and Hemophilus strains and almost inactive Staphylococci and common Gram-negative bacteria, e.g. Against Proteus, Escherichia and Pseudomonas.

Antibiotic SB 22484 is prepared by fermentation using a strain isolated from a soil sample collected in Italy and July 1983.

We deposited on the 6th in the collection of an international depository body, the Agricultural Research Collection (NRRL, Peoria, Illinois, United States), in accordance with the provisions of the Budapest Treaty. The tribe is

It is assigned a deposit number NRRL 15496.

Streptomyces sp. The characterization of NRRL 15496 is given below.

Macroscopic and microscopic examination

The colonies are characterized by a yellow to deep chromium yellow vegetative mycelium, which is formed on most of the media examined, and a turquoise green mycelium is observed. The spore carriers in spirals, respectively. reel or hook shapes are located on a bar. The spores are cylindrical with rounded ends; their diameter is 1.2 x 2-3 µm.

Based on the shape of the spore carriers and the color of the aerial mycelium, Streptomyces sp. NRRL 15496 is classified in the "spira-green class", Pridham

Hesseltine and Benedict (T.G. Pridham. C.W. Hesseltine and R.G. Benedict: A Guide for the classification of streptomycetes according to selected groups. Appl. Microb. Vol. 6, 1958).

Characteristics of colonies Characteristics of the cultures

To study the characteristics of the cultures, Streptomyces sp. NRRL 15496 was cultured on various standard media according to Shirling and Gottlieb (1), with the addition of several media recommended by Waksman (2).

The color, if necessary, is determined according to the method of Maerz and Paul (3).

Observations shown in Table 1 are performed after seven weeks of incubation at 28 ° C.

The pH of the media used for the classification is approx. neutral (pH 6.6-7).

Streptomyces sp. Characteristics of cultures of strain NRRL 15496

broth

Vegetative mic.

Aerial mycelium.

Soluble pigment

Oatmeal Agar

ISP # 2 broth

Oatmeal Agar / ISP No. 3 medium rich growth, thick, wrinkled surface, yellowish-green

9 / L / 6 plentiful growth, wrinkled surface, chrome yellow 9 / L / 7 plump growth, smooth surface, golden-yellow 9 / K / 6 yellow, turquoise spores yellow, / sparse sporake'pzes / turquoise

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broth

Table I (cont'd)

Vegetative mic. Aerial mycelium.

Soluble pigment

Hardening Agar / ISP No. 4

Glycerol-Asparagine Agar / ISP No. 6 media /

Peptone Yeast Extract Iron Agar / ISP

No. 6 medium / Tyrosine agar / ISP

No. 7 medium / Hickey-Tresner agar

Bennet agar

Nutrient Agar

Abundant growth of potato agar, turquoise smooth surface, cream spores, moderate formation abundant growth, thick, smooth surface, lemon yellow

9 / J / 2 moderate growth, smooth surface, colorless abundant growth, smooth surface, light hazelnut abundant growth, turquoise green wrinkled surface, spores of ga, green patches moderate growth, 9/1/3 formation moderate growth, wrinkled surface , chrome lemon,

9/1/3 moderate growth, smooth surface, light cream abundant growth, turquoise wrinkled surface, deep spores, sparse chrome, 9/6/7 formation

Peptone-Glucose generous growth, - - agar slightly wrinkled, worldly gos cream Czapek glucose generous growth, traces - agar wrinkled and crusty white air surface, yellow mycelium 9/1/5 Czapek sucrose sparse growth, - - agar thin surface Ca-malat agar moderate growth, - - thin surface, ka- yellow 10/1/1 skimmed milk bushy growth, - - wrinkled surface, bo- rostyánsárga ovalbumin sparse growth, - -

straw yellow / 1 / E.B. Shirling, D. Gottlieb, 1966: Method tor characterization of Streptomyces species. Int. J. Syst. Bacteriology. 16, 313-340.

/ 2 / S.A. Waksman, 1961: The Actinomycetes. Vol 2., 328-334. The Williams and Wilkins Co., Baltimore.

/ 3 / A. Maerz, M. Reá Paul, 1950: A Dictionary of Color. 2 ⁿ Ed., McGraw-Hill Book Company Inc., New York.

Coal resources - by Shirling and Gottlieb

Streptomyces sp. We tested the method described in NRRL 15496 - Part II. θ®, which aims to exploit various carbon sources.

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II. spreadsheet

Carbon utilization

inositol - fructose +++ rhamnose +++ mannitol +++ xylose +++ raffinose - arabinose ++ cellulose - saccharose - mannose + ~ H + lactose +++ galactose +++ salicin + dextrose ++ ++ intensive recovery ++ moderate + weak - does not utilize Physiological characteristics In III. Table a Streptomyces sp. NRRL 15496 describes physiological characteristics. III. spreadsheet Physiological characteristics test Results Hardening hydrolol taste +++ H ₂ S formation + formation of melanin - tyrosine hydrolysis + hydrolysis of casein calcium malate hydrolysis - nitrate reduction litmus milk no coagulation no peptonization gelatin disbursement ++ +

- negative response + weak positive response + + positive response + + + strongly positive response ± negative or weak positive response

A species of Streptomyces capable of producing the antibiotic SB 22484, such as Streptomyces sp. NRRL 15496 or an antibiotic-producing variant or mutant thereof, SB 22484, is cultured aerobically in an aqueous medium containing an assimilable carbon source, an assimilable nitrogen source, and inorganic salts. This medium may be identical to one of the many media normally used in the fermentation area, but certain media are preferred. so e.g. preferred sources of carbon include glucose, mannose, galactose, starch, corn flour, and the like. Preferred sources of nitrogen are ammonia, nitrates, soybean meal, 4 peptones, meat extracts, yeast extracts, tryptone, amino acids and the like. The inorganic salt components of the medium may be selected from the conventional salts which are soluble and capable of sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulfate, phosphate, nitrate, and the like. release of ions.

The antibiotic-producing strain is usually pre-cultured in a shake flask and this culture is used to inoculate smaller glass fermenters to produce more substantial amounts of the antibiotic. The medium used for pre-culture may be the same as that used for larger fermentations, but other media may also be used. SB 22484 producing strains were ca. 20 ° C and approx. It can be cultured at a temperature of 40 ° C, preferably about 40 ° C. 24-30 ° C.

During the fermentation, antibiotic production can be monitored by examining the antibiotic activity of the samples of the fermentation broth or mycelium extract, e.g. by biological assays or by TLC (HPLC) or HPLC (high performance liquid chromatography).

For this purpose, organisms known to be sensitive to antibiotics SB 22484 may be used. A particularly useful test organism is Neisseria caviae ATCC 14659 and Streptococcus dysgalactiae ATCC 9926. Biological assays are preferably performed by agar diffusion on agar plates. The maximum antibiotic production is usually fermentation

Performs between 2nd and 5th day.

Streptomyces sp. The antibiotic SB 22484 produced during fermentation with strain NRRL 15496 is mainly found in the fermentation broth. Therefore, the recovery of the antibiotics is preferably accomplished by extraction of the filtered fermentation broth. Extraction of the filtered fermentation broth is particularly preferably with ethyl acetate, but other lower alkyl alkanoyl esters which are immiscible with water, such as, for example, ethyl acetate, are suitable. methyl acetate, ethyl propionate and butyl propionate; and lower hydrocarbons such as polyhalogenated hydrocarbons, e.g. methylene chloride, dichloroethane and chloroform, and lower alkyl ketones, e.g. methyl isopropyl ketone, methyl isobutyl ketone, higher alkanols, e.g. butanol, pentanol and cyclohexanol, etc. too.

The crude antibiotic SB 22484 is then recovered from the extraction solvent by conventional means; such as preferably concentrating the extraction solvent to a small volume and precipitating the crude antibiotic from this solution by adding a non-antibiotic precipitant.

The purification of the crude antibiotic can then be accomplished by a variety of methods, e.g. column chromatography, salt formation or extraction with a suitable solvent. If purification is by column chromatography,

-4193696 is preferably the so-called. spherical exclusion chromatography is used to obtain optimum separation results. Specifically, Sephadex LH-20 (Pharmacia Fine Chemicals), a controlled pore size cross-linked dextran in which most hydroxyl groups are alkylated, has proved to be an exceptionally useful stationary phase and provides excellent purification. Alternatively, the filtered fermentation broth may be adjusted to pH 5-9 and a functionally unmodified polystyrene resin such as Amberlite X-AD-2 (or X-AD-4, X-AD-7, X-AD-8). or Diaion HP 20 resin column, eluting with deionized water, using a binary mixture of 10% to 90% aqueous acetone, methanol, ethanol, n-propanol, isopropanol or tetrahydrofuran. Fractions were pooled according to the antibiotic content, evaporated to dryness, adjusted to pH 4-8 and extracted as described above for the filtered fermentation broth.

According to another preferred method, the pH of the filtered fermentation broth is about. It is adjusted to 7.5 and extracted with a water immiscible lower alkyl alkanoyl ester, preferably butyl acetate. The organic layer was concentrated and its pH was ca. Set to 9.0. This pH adjustment is preferably achieved by the addition of sodium diethyl malonate. The precipitate was taken up in water and its pH was ca. 4 and extracted again with the selected water-immiscible lower alkyl alkanoyl ester. The pH is ca. By adjusting it to 9, the precipitation is repeated with the addition of a base, preferably sodium diethyl malonate. Thus, a purified composition is prepared containing the antibiotic of the invention. Other column chromatography techniques such as adsorption chromatography or fractional chromatography may also be used for this purification.

Purification by salt formation is simply accomplished by dissolving the antibiotic in a suitable solvent and adding an equimolecular amount of base. The salt formed is precipitated by the addition of a non-soluble precipitant and recovered by filtration. The free acid antibiotic is prepared by dissolving the crude antibiotic in ethyl acetate and treating the organic solution with an acidic buffer, preferably a phosphate buffer, for approx. Extract at pH 4.0.

The product is then recovered essentially using conventional techniques, e.g. by precipitation from the organic layer or by concentrating the mixture to a small volume. If the potency of the product is not high enough, it can be further purified by column chromatography, essentially according to the same procedure as described above for the first purification.

If purification by column chromatography is used, the entire purification procedure can be followed by HPLC.

Fractions of similar HPLC profile were combined and evaporated to dryness. This gives essentially pure antibiotic SB 22484.

Physiochemical properties of antibiotic SB 22484

The antibiotic SB 22484 has the following properties:

A) UV absorption spectrum as shown in Figure 1 showing the following absorption maxima:

λ _max (nm) λ _{cm -1} a) 0.1 N in HCl

230 283 332 663 192 343 b) pH 7.38 in phosphate buffer 230 719 323 391 c) 0.1N sodium hydroxide 323 387 d) methanol 229 715 320 165 B) IR absorption spectrum as shown in FIG. according to Figure

showing the following absorption maxima (cm ^-1 ):

3700-3080, 2980-2840 (nujol); 1645; 1560;

1455 (nujol); 1375 (nujol); 1305; 1240;

1215; 1145; 1090; 1060; 1035; 990; 945; 910;

890; 850; 810; 720 (nujol)

C) NMR spectrum as shown in Figure 3 showing the following signal groups (in ppm) in hexadeuteroaceton (270 MHz, 1H-NMR) using TMS (0.00 ppm) as internal standard;

0.77 (3H), 0.83 (3H), 1.21-1.24 (3H), 1.2-2.7 (5H), 1.67 (s, 3H), 1.73 (3H) ), 2.01 (s, 3H), 3.20 (s, 3H), 3.3-5.6 (UH), 5.4-6.7 (13H), 6.04 (1H), 7 , 40 (1H), 7.68 (1H), 10.53 (1H) (s = singlet)

D) Mass spectra recorded by LC-MS (Direct Liquid Chromatography Mass Spectroscopy) using an HP 5985B instrument in a negative ion mode with LC under the following conditions:

column: Brownlee Foot. RP RP 8-10 pm, cm, flow rate: 1 ml / min, eluent; 60:40 acetonitrile: 0.1M ammonium formate. The spectrum shows 4 major peaks with a retention time of 4.48, 5.03, 5.65 and 53.5, respectively. 7.25 minutes and is conditionally referred to as antibiotic SB 22484 Factors 1, 2, 3 and 4.

The major fragmentation peaks for the above antibiotic factors, i.e. antibiotics SB 22494 Factors 1, 2, 3 and 4, are as follows:

Factor 1: 752, 734, 716, 684, 538, 598, 566, 500

Factor 2: 766, 748, 730, 698, 652, 612, 580

Factor 3: 752, 734, 716, 684, 638, 598, 566, 500

Factor 4: 766, 748, 730, 698, 652, 612, 580, 500

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E) The following Rf values are obtained using silica gel 60 F ₂₅₄ Merck plates silanized on various chromatographic systems:

Elution System / v / v / v / Rf value 1) NaH ₂ PO ₄ / methanol 4/6 ..... 0.44 0.50 0.55 2) NaH ₂ PO ₄ / methanol / acetonitrile 4/5/1 0.52 0.57 0.63 3) 0.05M tetrabutylammonium Phosphate (pH 7.0): methanol 4/6 0.46 0.50 0.55

Detection: UV light at 254 nm

The antibiotic SB 22484, as an antibacterial agent, is particularly active against bacterial strains responsible for infectious diseases which are generally difficult to cure (such as gonorrhea and meningitis). As mentioned above, the antibiotic of the invention is active against Neisseria strains, sufficiently active against Steptococcus, Ureaplasma, Mycoplasma and Hemophylus strains, while it is almost inactive against Staphylococci and common Gram-negative bacteria such as Proteus, Escherichia and Pseudomonas.

The antibacterial activity of the compounds of the invention can be detected in vitro by a standard dilution assay.

Iso-Sensitest broth (Oxoid) is used for Staphylococci, and Todd-Hewitt broth (Difco) is used for Streptococci; Neisseria strains were grown on OC agar containing 1% Iso-Vitalex (BBL) (Difco) and Hemophylus strains were grown on OC agar containing 1% Iso-Vitalex (BBL) and 1% hemoglobin (Difco) (DIFCO) . The broth cultures were diluted so that the final inoculum was ca. 1Ο ⁴ -10 ³ colony forming units per millimeter (CFU / ml). Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of antibiotic which, after incubation for 18-24 hours at 37 ° C, eliminates visible growth. Neisseria strains were cultured in CO ₂ rich atmosphere.

The in vitro activity spectrum of antibiotic SB 22484 is summarized in the following table.

ARC. spreadsheet

In vitro antibacterial activity of SB 22484 strain_MIC (pg / ml)

S. pyogenes C 203 SKF 13400 8

S. dysgalactiae ATCC 9926 8

S. pneumoniae UC 41 L 44 4

H. influenzae ATCC 9795 b type 32

Strain MIC (gg / ml)

H. influenzae ATCC 9332 d type 32

H. influenzae L 990 ⁺ 32

M. gallisepticum 9/6 Weybridge L 431 4

N. caviae ATCC 14659 0.5

N. gonorrhoeae NCTC 8254 0.5

N. gonorrhoeae L 997+ 2

N. gonorrhoeae ⁺ L 1596 ⁺⁺ 2

N. gonorrhoeae ⁺ L 1599 ⁺⁺⁺ 1

N. meningitis ATCC 13077 (A) 4

N. meningitis ATCC 13090 (Β) 1

N. meningitis ATCC 13102 (C) 2

N. meningitis ATCC 13118 (D) 1

N. meningitis ATCC 13804 1

The L symbol refers to the applicant's own collection number.

⁺ Clinically isolated strains identified by the applicant's own collection number.

⁺⁺ Clinically isolated strains resistant to Spectinomycin (MIC above 128 in these conditions).

⁺⁺⁺ Clinically isolated strains producing penicillinase beta (resistant to penicillin with MICs above 50 under these conditions).

The activity of Ureaplasma urealyticum is assayed by pre-culturing the microorganism in the following medium:

PPLO broth without crystal violet (Difco) 80 ml

IN HCl 0.9 ml horse serum (Sclavo) 10 ml

50% urea (C. Erba RP) in 1 mL

0.2% phenol red 1 ml approx. Adjusted to pH 6.

The minimum inhibitory concentration (MIC) was determined in test tubes by the double batch dilution method (concentration range: 0.008 to 128 pg / ml).

Volumes of 1 ml per tube were inoculated with 10 ^{2 to} 10 ³ color changing units (ccu) from 20-24Vs broth culture. The tubes were incubated at 37 ° C.

The MIC endpoint is defined as the lowest concentration of antibiotic that prevents discolouration, as measured by the time at which the control culture shows a discoloration at pH 7.8 (determined by comparing the culture color to the same medium adjusted to different pH values). color). This change takes approx. ^It occurs after 24 ^A incubation if ca. Inocula with 10 ² -10 ³ ccu / ml were used.

Inoculum size was determined as follows: 10-fold serial dilutions of broth cultures used as inoculum were added to broth; ccu / ml is calculated from the lowest dilution showing color change.

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The results of this experiment for a clinically isolated strain are shown below:

MIC (pg / ml)

U. urealyticum L 1479 8

In in vitro bactericidal assays, antibiotic SB 22484 kills 99.9% of Neisseria gonorrheae L 997 cells in logarithmic growth phase when cultured on GC agar medium (Difco) containing 1% Iso-Vitalex (BBL) for 24 hours, After incubation at 37 ° C, at 5 times the MIC (10 pg / ml).

Preliminary in vivo studies show that antibiotic SB 22484, administered at 200 mg / kg sc, to mice infected with S. pyogenesis - according to V. Arioli et al., J. Antibiotics 29, 511, 1976 - 3 of 5 infected animals protect. Furthermore, it is noteworthy that the compounds of the present invention show very low toxicity, since their LD ₅₀ values are greater than 1000 mg / kg (the highest dose tested) in mice administered ip.

Due to their antimicrobial activity, the compounds of the present invention are useful as active components of antimicrobial compositions used in human and veterinary medicine for the prophylaxis or treatment of infectious diseases caused by susceptible pathogenic bacteria.

Due to their antimicrobial activity, antibiotic SB 22484 and the corresponding salts can be used in human or veterinary medicine. In particular, antibiotic SB 22484 and the corresponding non-toxic pharmaceutically acceptable salts can be used in any ratio.

As noted above, antibiotic SB consists of at least 4 major components (antibiotic SB 22494 Factors 1, 2, 3 and 4). As will be appreciated by those skilled in the art, the percentages of these factors in the complex may vary from experiment to experiment, depending on various parameters such as culture medium and fermentation conditions, and routine changes in the isolation and purification process. The description of the invention therefore extends to L, 2,

Factors 3 and 4 for any proportion of antibiotic SB 22484 blends. These mixtures are generally referred to as antibiotic SB 22484.

Due to its antimicrobial activity, antibiotic SB 22484 is the most suitable antibiotic for the treatment of gonorrhea. Gonorrhea is currently treated with a number of different antibiotics - primarily penicillin and spectinomycin, or tetracycline, and the like. ampicillin - treated. However, given that the occurrence of gonorrhea is the latter

Increased steadily in 15 to 20 years, the widespread use of these antibiotics in the treatment of gonorrhea has resulted in an increasing frequency of drug resistance. against resistant strains - represents progress in the treatment of this disease.

For antibiotic treatment, antibiotic SB 22484 and its non-toxic pharmaceutically acceptable salts can be administered in various ways, e.g. topical or parenteral administration. However, the preferred route of administration is parenteral administration.

Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or flavoring vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

Alternatively, the active ingredient may be in powder form and may be formulated at the time of use when a suitable vehicle, e. sterile pro inj. water is added to the powder.

Depending on the mode of administration, these compounds may be formulated into various dosage forms.

In some cases, the compounds of the invention may be formulated in enteric-coated dosage forms for oral administration by methods known to those of ordinary skill in the art (e.g., Remington's Pharmaceutical Sciences, 15 ^/ Ed., Mack Publ. Co., Easton, Pennsylvania, USA, p.1614).

This is particularly the case when it is desirable for the antimicrobial agent to be particularly active or adsorbed in the intestinal tract, while still passing through the gastrointestinal tract.

The amount of active ingredient to be used will depend on various factors, such as the weight and condition of the individual being treated, the mode and frequency of administration, and the trigger involved.

The antibiotic of the invention, namely the antibiotic SB 22484, and physiologically acceptable salts thereof, are generally effective at ca. 5- kb At a daily dose of 100 mg of active component / kg of body weight, divided into 2 to 4 times daily dosing.

Particularly preferred are those formulations which have been prepared for about one hour. 100 - approx. They are prepared in unit dosage form containing 5000 mg / unit of active ingredient.

However, when used in the therapy of gonorrhea, where single dose therapy is highly preferred for practical reasons, higher minimum doses of antibiotic SB 22484, typically 10-100 mg / kg, should be used to ensure that the

-7193696 Maintain effective blood levels for an extended period of time.

In addition, in the treatment of gonorrhea, a sustained release parenteral dosage form is preferably formulated. Formulations of delayed action may be prepared according to a variety of mechanisms and methods known to those skilled in the art. According to a preferred method, the preparation of sustained release formulations containing antibiotic SB 22484 comprises a water-insoluble salt of this antibiotic, e.g. benzathine, procaine, hydrabamine, etc. salt - used in suspension in an aqueous or oily medium.

These salts, i.m. when ingested, they are actually released very slowly due to their low water solubility and thus result in sustained blood levels of the antibiotic.

Manufacture of pharmaceutical preparations

L. M. A unit dosage form for injection is prepared by suspending 2000 mg of the prodrug salt of antibiotic SB 22484 in 3 ml of refined peanut oil, which is solidified with 2% aluminium stearate.

L. M. A unit dosage form for injection is prepared by dissolving 250 mg of antibiotic SB 22484 in 5 ml of the following composition: 40% propylene glycol, 10% ethanol, 0.5% (v / v) trihydroxymethylamine. 50%.

L. M. unit dosage form for injection using 5 ml of sterile USP solution containing 8% propylene glycol and 3500 mg of antibiotic SB 22484.

Intramuscularly for injection, a unit dosage form is prepared such that 2000 mg of the sodium salt of antibiotic SB 22484 is prepared in 5 ml of sterile pro inj. suspended in water.

The following examples are provided to illustrate the invention in more detail, but are not to be construed as limiting the scope of the invention.

Example 1. Preparation of the antibiotic

A lyophilized test tube was opened containing Streptomyces sp. NRRL 15496 and transferred to sterile oatmeal agar tube under sterile conditions. 10 daily, approx. After incubation at 28 ° C, the slanted culture was suspended in distilled water and a 500-ml baffle was inoculated into Erlenmeyer flasks containing 100 ml of the following medium:

beef extract 5 g peptone 5 g enzymatic casein hydrolyzate 3 g yeast extract 5 g

NaCl 1.5 g dextrose 20 g distilled water 1000 ml.

The flasks were stored at 28 ° C for approx. Incubate for 48 hours at 220 rpm.

The contents of the flask are used to inoculate a 10 liter fermenter containing 6 liters of fermentation broth of the following composition:

beef extract 4 g peptone 4 g

NaCl 2.5 g yeast extract 1 g soy meal 10 g dextrose 25 g

CaCO ₃ 5 g tap water 1000 ml.

The fermentation was carried out at 28 ° C at 900 rpm; the aeration rate is 1 vol / vol / min.

The maximum antibiotic activity is approx. It is reached after 40 hours of fermentation.

Antibiotic levels were determined using a paper disk agar plate assay using Neisseria caviae ATCC 14659 and Streptococcus dysgalactiae ATCC 9926 as the test organism.

Example 2. Production of antibiotic in large quantities

A Streptomyces sp. The lyophilized test tube containing the NRRL 15496 culture and its contents are transferred under sterile conditions to an inclined tube containing oatmeal agar. After incubation for 10 days at 28 ° C, the slant culture was resuspended in distilled water and the suspension was 2 x. Inoculate a 2000 ml Erlenmeyer flask with a baffle plate containing 500 ml of medium containing:

meat extract 5 g peptone 5 g enzymatic casein hydrolyzate 3 g yeast extract 5 g

NaCl 1.5 g dextrose 20 g distilled water 1000 ml

These two flasks were incubated at 28 ° C at 220 rpm for approx. 40 hours.

The culture in the two flasks is used to inoculate a 50 liter fermenter containing 30 liters of inoculum medium with the following composition:

beef extract 4 g peptone 4 g

NaCl 2.5 g yeast extract 1 g soy meal 10 g dextrose 25 g

CaCO ₃ 5 g

CaCO ₃ 5 g tap water 1000 ml.

The fermentation was carried out at 28 ° C at 500 rpm; the aeration rate is 1 vol / vol / min.

After 1 hour of fermentation, a 300 liter fermenter was inoculated with 30 liters of inoculum culture and 200 liters of the same

Contains -8193696 medium. This fermenter was incubated at 28 ° C at 240 rpm with an aeration rate of 1 v / v.

The maximum antibiotic activity is approx. It is reached after 40 hours of fermentation.

Antibiotic levels were determined using a paper-disk agar plate assay using Neisseria caviae ATCC 14659 as an experimental organism previously cultured overnight at 37 ° C in a cerebral heart agar medium. The culture results in a 23 cm inhibition zone on a 10 cm paper plate.

Example 3. Isolation and purification of antibiotic SB 22484

The 60 ml fermentation broth is filtered in the presence of Clarcel ^R , a filtration aid, and the pH of the filtrate is ca. Adjust to 7.0 and extract with ethyl acetate (about 60 mL). The organic phase was separated by centrifugation and concentrated to a small volume. Petroleum ether was added to the oily residue and the precipitated crude antibiotic was collected by filtration. A portion (4.5 g) of this material is dissolved in 50 ml of methanol, applied to a 1 m chromatography column containing 1.5 ml of Sephadex L20 suspended in methanol and the system is extracted with the same solvent.

Fractions of about 20 ml were collected and their activity determined by paper disk diffusion assay using N. caviae ATCC 14659 and by TLC and HPLC analysis.

The antibiotic-containing fractions were combined and evaporated to dryness. The oily residue was dissolved in a minimum amount of tetrahydrofuran, ethyl ether was added and the mixture was shaken until a precipitate formed. This was collected and dried. About 960 mg of pure preparation of antibiotic SB 22484 having the above physiochemical properties are obtained.

Claims (5)

PATENT CLAIMS 1) NaH ₂ PO ₄ / methanol 4/6 = 0.44 0.50 0.55 Factor 1: 752, 734, 716, 684, 538, 598, 566, 500 A process for the preparation of the novel antibiotic SB 22484 complex, the major components of which are antibiotics SB 22484 Factors 1, 2, 3 and 4, having the following properties: A) UV absorption spectrum as follows with adsorption maxima: larnbda _mox (nm) L-i Ci a) in 0.1N HCl 230 663 283 192 332 343 b) pH 7.38 in phosphate buffer 230 719 323 391 c) 0.1N sodium hydroxide 323 387 d) methanol 229,715 320 365 B) IR adsorption spectrum, showing the following adsorption maxima (cm- ¹ ): 3700-3080, 2980-2840 (nujol); 1645; 1560; 1455 (nujol); 1375 (nujol); 1305; 1240; 1215; 1145; 1090; 1060; 1035; 990; 945; 910; 890; 850; 810; 720 (nujol), C) The NMR spectrum shows the following signal groups (in ppm) in 270 MHz 'H-NMR mode in hexadeuteroacetone using TMS (0.00 ppm) as internal standard in ppm: 0.77 (3H), 0.83 (3H), 1.21-1.24 (3H), 1.2-2.7 (5H), 1.67 (s, 3H), 1.73 (3H) ), 2.01 (s, 3H), 3.20 (s, 3H), 3.3-5.6 (UH), 5.4-6.7 (13H), 6.04 (1H), 7 , 40 (1H), 7.68 (1H), 10.53 (1H), D) mass spectrum recorded by direct liquid injection LC-MS using an HP 5985 B instrument operating in the negative ion mode and performing the LC under the following conditions: column: Brownlee Foot. Rp 8 10 µm, 25 cm (silica gel functionalized with octylsilane), flow rate: 1 ml / min, eluent: acetonitrile: 0.1 M ammonium formate 60:40, showing four major peaks with a retention time of 4.48, 5.03, 5.65 ill. 7.25 minutes, with corresponding components called antibiotic SB 22484 L, Factors 2, 3 and 4, E) the following major fragmentation peaks for the antibiotic factors mentioned above, namely antibiotics SB 22484, 1,2, 3 and 4: The method of claim 1, wherein the production strain is cultured at a temperature of 24-30 ° C. 2) NaH ₂ PO ₄ (methanol) acetonitrile Factor 2: 766, 748, 730, 698, 652, 612, 580 3. A process according to claim 1, wherein the filtered fermentation broth is a water-immiscible (C1-C6) alkyl (C2-C6) alkanoic acid18 for recovering the antibiotic. ester, C 1-4 polyhalogenated hydrocarbon or C 4-7 alkanol. 3) 0.05M tetrabutylammonium phosphate (pH 7.0): methanol 4/6 mixture 0.46 0.50 0.55 detection with UV light (254 nm), -9193696, characterized in that Streptomyces sp. The strain NRRL 15496 or an antibiotic-producing variant or mutant thereof, SB 22484, is cultured under immersed aerobic fermentation conditions in the presence of assimilable carbon and nitrogen sources and inorganic salts, and the said antibiotic is recovered from the fermentation broth and isolated. Factor 3: 752, 734, 716, 684, 638, 598, 566, 500 4. The process of claim 1, wherein the antibiotic is isolated by steric exclusion column chromatography using a controlled pore size cross-linked dextran derivative or a functionally unmodified polystyrene. The 4/5/1 mixture is 0.52 0.57 0.63 Factor 4: 766, 748, 730, 698, 652, 612, 580, 500 (F) the following R / values for various chromatographic systems using silanized silica gel 60 F ₂₅₄ Merck plates: elution system / volume / volume / volume / Value 5. The method of claim 4, wherein the chromatography column is eluted with 10% to 90% aqueous acetone, methanol, ethanol, n-propanol, isopropanol or tetra15 hydrofuran.